DNA methylation biomarkers for early detection of gastric and colorectal cancers

Cancer is one of the leading causes of death worldwide. The early diagnosis of cancer remains one of the greatest cancer research challenges. Epigenetic alterations, such as altered DNA methylation, that occur during the early stages of car­ cinogenesis have been proposed as candidate cancer biomark­ ers. In recent years detection of small amounts of methylated DNA in samples, including blood and stool, has demonstrated the feasibility of DNA methylation as a molecular cancer bio­ marker. The translational promise of aberrant DNA methy­ lation includes screening and detecting cancer, evaluating prognosis, assessing treatment efficacy, and detecting minimal residual disease ( Figure 1 ). The application of DNA methyl­ ation biomarkers for cancer detection has been studied most intensively. Alterations in DNA methylation patterns in the genome have been observed across malignancies and usually occur before other detectable genetic changes 1 . Therefore, biomarker mining for the early diagnosis of cancer based on DNA methylation has emerged as a promising field and has become a focus of research globally. Although hundreds of DNA methylation biomarkers have displayed great poten­ tial for early cancer detection, only a few methylation bio­ markers have been used in the clinical setting to date. The National Medical Products Administration (NMPA) in China has approved 20 methylation­based commercial kits for can­ cer diagnosis. More than one­half of these kits are used for colorectal cancer (CRC) diagnosis (11); one kit is used for gastric cancer, three for cervical cancer, two for lung cancer,

Cancer is one of the leading causes of death worldwide.The early diagnosis of cancer remains one of the greatest cancer research challenges.Epigenetic alterations, such as altered DNA methylation, that occur during the early stages of car cinogenesis have been proposed as candidate cancer biomark ers.In recent years detection of small amounts of methylated DNA in samples, including blood and stool, has demonstrated the feasibility of DNA methylation as a molecular cancer bio marker.The translational promise of aberrant DNA methy lation includes screening and detecting cancer, evaluating prognosis, assessing treatment efficacy, and detecting minimal residual disease (Figure 1).The application of DNA methyl ation biomarkers for cancer detection has been studied most intensively.Alterations in DNA methylation patterns in the genome have been observed across malignancies and usually occur before other detectable genetic changes 1 .Therefore, biomarker mining for the early diagnosis of cancer based on DNA methylation has emerged as a promising field and has become a focus of research globally.Although hundreds of DNA methylation biomarkers have displayed great poten tial for early cancer detection, only a few methylation bio markers have been used in the clinical setting to date.The National Medical Products Administration (NMPA) in China has approved 20 methylationbased commercial kits for can cer diagnosis.More than onehalf of these kits are used for colorectal cancer (CRC) diagnosis (11); one kit is used for gastric cancer, three for cervical cancer, two for lung cancer, and the remaining three are used for the diagnosis of gliomas, and liver and bladder cancers.In the US, seven DNA methyla tionbased assays are available commercially to help clinicians make better treatment decisions in patients with cancer 2 .Two assays can be used to detect CRC and one can be used to detect > 50 types of tumors.
Unlike Western countries, gastric cancer and CRC are highly prevalent in China with > 480,000 patientrelated deaths, accounting for 20.1% of all cancerrelated deaths 3 .The inci dence of CRC in China has rapidly increased.CRC currently ranks second with respect to morbidity among all malignancies 3 .The incidence of gastric cancer in China is among the high est worldwide, accounting for > 45% of all new gastric cancer cases 3 .Gastric cancer and CRC have a poor prognosis and are difficult to diagnose in the early stages due to a lack of charac teristic clinical manifestations.In highrisk groups, endoscopy with tissue biopsies is the gold standard for diagnosing gastric cancer and CRC; however, endoscopy is invasive and highly dependent on the judgment and experience of the endoscopic specialist.Unfortunately, the currently available protein mark ers, such as CEA, CA199 and CA724, are ineffective in detect ing earlystage gastrointestinal cancer owing to a low sensitivity.There is an ongoing quest for reliable non invasive biomark ers with better sensitivity and specificity for the detection of gastrointestinal cancer to complement the currently available screening methods.Gastric cancer and CRC share many bio logical features.For example, both stomach and colorectum epithelia are derived from endoderm.Normal cells undergo a hyperplasianeoplasiacancerous process during tumorigenesis to become cancerous.Notably, gastric cancer and CRC share many aberrant DNA methylations, including SEPT9, MGMT, and SDC2.Therefore, in this perspective we focused on the pro gress in research involving DNA methylationbased diagnostics for gastric cancer and CRC screening and early detection.

Colorectal cancer
Screening for CRC using a fecal immunochemical test (FIT) has been shown to reduce CRCrelated mortality; however, a FIT is limited by relatively low specificity and sensitivity for early CRC detection.Recently, several methylated genes have been studied epigenetically as alternative biomarkers to FIT.

Blood-based DNA methylation biomarkers for screening and early detection of CRC
To date, several potential bloodbased DNA methylation biomarkers have been identified for CRC detection, includ ing BCAT1, BMP3, C9orf50, CDKN2A, CLIP4, KCNQ5, MLH1, NDRG4, PRIMA1, SDC2, SEPT9, SFRP2, and VIM 2,4 (Table 1).In fact, the bestknown blood epigenetic marker for CRC is SEPT9.Methylated SEPT9 is the only single gene methylation biomarker approved by the U.S. Food & Drug Administration (FDA) for CRC detection, as well as the first methylation biomarker approved by the NMPA in China.Methylation changes in SEPT9, a member of the septin family, which is involved in cytokinesis and cytoskel etal organization, have been linked to multiple cancers.In casecontrol and opportunistic screening studies, plasma methylated SEPT9 demonstrated approximately 70% sensi tivity and 90% specificity for detecting CRC 4 .In a large pro spective CRC screening cohort, the sensitivity and specificity of methylated SEPT9 were estimated to be 48.2% and 91.5%, respectively 5 .Furthermore, among patients with TNM and Duke stage progression, the positive methylated SEPT9 rates gradually increase 6 .Of note, the criteria for determining methylated SEPT9 positivity vary across studies.For exam ple, in some studies a positive reaction was indicated by a methylated SEPT9 curve exceeding the prespecified thresh old of 50 polymerase chain reaction (PCR) cycles 5 , whereas a predetermined threshold of 45 PCR cycles was applied in other studies 7 .In addition, there is inconsistency in the PCR repeat systems used across different studies; most studies use triplicate PCR reactions, while other studies use double rep licates 5 .Therefore, the methylated SEPT9 test performance across studies may reflect differences in the study popula tions, different interpretation thresholds among commer cially available kits, and differences between study settings (retrospective casecontrol study vs. opportunistic vs. popu lationbased screening).

Combined detection of multiple targets
Although singlegene methylation biomarkers have demon strated promising specificity for CRC, the sensitivity is insuf ficient.Therefore, multigene combined testing, which has attracted much attention in recent years, may improve the sensitivity of CRC detection.Imperiale et al. 11 proposed the use of FIT in addition to assessing KRAS mutations, aber rant NDRG4, and BMP3 methylation for the early detection of CRC in stool samples.FIT demonstrated a 73.8% sensitiv ity and 94.9% specificity when used independently in CRC detection, and a 92.3% sensitivity and 86.6% specificity when combined with DNA testing 11 .Although the sensitivity of the multitarget stool DNA test did not vary significantly according

Strengths and weaknesses between blood-and stool-based DNA methylation biomarkers for CRC detection
No headtohead studies have compared the efficacy of these commercially available methylated gene detection kits in the same patient cohort.Based on studies with small sam ple sizes, methylated gene detection in stool samples did not demonstrate superiority over the detection of the same genes in plasma samples (Table 1).Of note, a blood sample can be obtained safely and objectively at any time, while a stool sam ple may not be collectible on demand.It is difficult to control feces quality and the characteristics of feces, such as loose or watery stools, may affect the test results.Moreover, fecal meth ylation testing cannot be used to monitor recurrence after sur gical resection.Notably, the methylation biomarker detection rate in advanced adenomas was relatively low whether serum, plasma, or feces was analyzed.Although several methylation detection kits have been approved by the NMPA in China, it is important to note that the kits are a supplement to colonos copy, not a replacement.

Gastric cancer
Although early screening for gastric cancer via gastroscopy may improve overall survival 12 , the availability of reliable, sim ple, and noninvasive screening tests is more limited than for CRC.Several studies have recently been conducted to identify DNA methylationbased biomarkers in the plasma, serum, gastric juice, and fecal samples for gastric cancer diagnosis, albeit with varying specificity and sensitivity 13 .Early detection and in vitro diagnostics for gastric cancer have yet to reach clinics en masse.

Blood-based DNA methylation biomarkers for screening and early detection of gastric cancer
Several potential bloodbased diagnostic methylation bio markers have been identified for gastric cancer detection, including C13orf18, DLEC1, FLNC, HODX10, MGMT, PCDH10, RNF180, RPRM, RPRML, RUNX3, SEPT9, SFRP2, SOX17, THBS1, UCHL1, and ZNF569 13,14 (Table 2).RNF180 is one of the ring finger protein genes involved in the deg radation of its substrates as an E3 ubiquitin ligase.Genes belonging to this family have been implicated in various biological processes, including cell growth, differentiation, and tumorigenesis 15 .Our previous study showed that the average methylation rate and methylated CpG sites within the RNF180 promoter region in tissues increased with the severity of gastric mucosal lesions 16,17 .Therefore, methyl ated RNF180 may serve as a candidate biomarker for gastric cancer.As mentioned earlier, methylated SEPT9 has been identified as a noninvasive diagnostic biomarker for CRC; however, methylated SEPT9 is not CRCspecific.Elevated levels of methylated SEPT9 have been observed in various cancers, with 48%-56% of gastric cancer patients also test ing positive for methylated SEPT9 18 .One study reported that the RS19 test is a new bloodbased methylation assay for early gastric cancer detection that combines two methylated genes (RNF180 and SEPT9) in a single reaction to improve the rate for earlystage gastric cancer and gastric dysplasia detection 14 .The RS19 test is an effective approach with good sensitivity (62.2%) and high specificity (84.8%) for detecting gastric cancer 14 .The plasma RS19 test has higher sensitivity than methylated SEPT9 or RNF180 alone in detecting gas tric cancer and gastric dysplasia 14 .This study had the largest reported sample size, exceeding 1000 cases 14 .The RS19 test is the first epigenetic biomarker approved by the NMPA in China for detecting gastric cancer and is commercially avail able.Currently, the authors are conducting a multicenter communitybased gastrointestinal cancer screening program using methylated RNF180, SEPT9, FIT, and Helicobacter pylori stool antigen (NCT05996458).In addition, another retrospective study presented a DNA methylationbased panel (ELMO1, ZNF569, and C13orf18) for distinguishing gastric cancer 19 .The study was limited by a relatively small sample size (36 patients with gastric cancer and 38 controls).It is anticipated that results from a larger study on screen ing, surveillance, or other intendeduse populations will provide additional confirmation.Ongoing clinical trials are currently exploring the performance of novel blood DNA methylationbased panels for gastric cancer diagnosis (clini cal trials.gov:NCT04511559, NCT04947995, NCT05224596, NCT05336058, NCT05347524, and NCT05668910; https:// www.chictr.org.cn/:ChiCTR2300075157).Additional methy lation kits for gastric cancer screening may become available for clinical use in the future.

Stool-based DNA methylation biomarkers for screening and early detection of gastric cancer
Unlike CRC, only a few studies have investigated stoolbased DNA methylation biomarkers for gastric cancer diagnosis (Table 2).Because the shedding of gastric tumor cells occurs in the upper gastrointestinal tract, tumor DNA passes through the intestines and is expelled from the body with feces after exposure to gastric acid, bile, and digestive enzymes.As a result, there is a minimal amount of tumor DNA available for testing in the stool.Existing studies have also shown that stool DNA methylationbased biomarkers do not exhibit good per formance in detecting gastric cancer.

Future developments and perspective
Although DNA methylation biomarkers outperform tra ditional markers, such as CEA, CA199, and CA125, in diagnosing earlystage gastric cancer and CRC, the overall sensitivity and specificity remain insufficient to fully meet the needs of cancer screening, especially for gastric cancer.Importantly, the impact of DNA methylation biomarker based screening on reducing the incidence and mortality of gastrointestinal cancer remains unclear.Another potential limitation of DNA methylation biomarkers for routine can cer screening is the higher cost.To address these challenges and needs, several considerations are essential.First, specific combination algorithms are needed to better consolidate existing DNA methylation biomarkers and traditional tumor markers to improve the sensitivity of early cancer detection.Second, the genome has approximately 28 million CpG sites, which have enormous potential for mining.Therefore, it is necessary to mine and integrate novel methylation bio markers as diagnostic targets using genomewide profiling.Third, large randomized controlled trials are needed to verify whether DNA methylation markerbased cancer screen ing can reduce the incidence and mortality of gastrointes tinal cancer.Concurrent health and economic evaluations during such trials are necessary to assess costeffectiveness.Fourth, there is a stepwise accumulation of DNA methyla tion of tumor suppressor genes from precancerstocancers. Understanding whether patients without neoplastic lesions who test positive for DNA methylation biomarkers have a higher risk of developing cancer than the general population is also crucial.Therefore, quantitative detection and dynamic observation of DNA methylation levels may be helpful for these patients to determine whether or not the lesion is malignant.Presently, all DNA methylation kits approved by the NMPA in China are used for the diagnosis of a single cancer type.The advantage of biomarkers for single cancer screening is the rel atively clear identification of the corresponding target lesion in patients with positive detection.Moreover, the sensitivity and specificity of a single cancer methylation gene for cancer detection are high and the cost is relatively low, which warrants further development.However, a drawback is that for whole body screening, multiple markers need testing with a substan tial increase in costs.Therefore, pancancer DNA methylation biomarkers are more suitable for individuals undergoing wholebody cancer screening.PATHFINDER evaluated a pancancer earlydetection blood test based on DNA methyla tion signatures 20 .The latest study supports the feasibility of this blood test for multicancer early detection 20 .Unfortunately, this pan cancer screening technique overlooks 80% of early stage tumors (stage III), indicating substantial room for improve ment in sensitivity 20 .The U.S. FDA has approved this test as a groundbreaking advance, marking the commencement of a new era of global early cancer screening.Given the rapid advances in sequencing and analytical and computational technologies, DNA methylation biomarkers are emerging as a significant advance in optimizing cancer screening.

Figure 1
Figure 1 Main technologies for DNA methylation detection and clinical applications.

Table 1
Overview of promising DNA methylation biomarkers used in the diagnosis of CRC and adenomas

based DNA methylation biomarkers for screening and early detection of CRC
10 however, a metaanal ysis involving 8 studies concluded unsatisfactory diagnostic performance of methylated VIM, with a sensitivity of 54.6% and a specificity of 88.5%9.Methylated SDC2 was the first stoolbased methylation assay for CRC detection approved by the NMPA in China.The sensitivity of methylated SDC2 in fecal DNA for CRC was 83.8%, 42.1% for advanced adenomas, and 87.0% for earlystage CRC (stage III)10.Methylated SDC2 appears to be the most accurate single gene among stool DNA methylation tests for detecting CRC based on a metaanalysis 9 , albeit largesample clinical trials are needed for further validation.

Table 1
11ntinuedto cancer stage or location within the colon, the sensitivity was relatively higher in distal advanced precancerous lesions than in proximal lesions (54.5% vs. 33.2%)11.This panel of mul titarget stool DNA tests has been approved by the U.S. FDA and the NMPA in China for CRC diagnosis.

Table 2
Overview of promising DNA methylation biomarkers used in the diagnosis of gastric cancer